Animal Development Chapter 47 1 mm human embryo at approximately 6–8 weeks after conception

An organism’s development Is determined by the genome of the zygote and by differences that arise between early embryonic cells Cell differentiation Is the specialization of cells in their structure and function Morphogenesis Is the process by which an animal takes shape

Fertilization

The main function of fertilization Is to bring the haploid nuclei of sperm and egg together to form a diploid zygote Contact of the sperm with the egg’s surface Initiates metabolic reactions within the egg that trigger the onset of embryonic development Two phenomena are important here Acrosomal reaction Cortical reaction

The Acrosomal Reaction Is triggered when the sperm meets the egg Releases hydrolytic enzymes that digest material surrounding the egg Gamete contact and/or fusion Depolarizes the egg cell membrane and sets up a fast block to polyspermy

Acrosomal Reaction in Sea Urchins Sperm nucleus Sperm plasma membrane Hydrolytic enzymes Cortical granule Cortical granule EGG CYTOPLASM Basal body (centriole) head Acrosomal process Actin Acrosome Jelly coat Egg plasma Vitelline layer Fused plasma membranes Perivitelline space Fertilization envelope Cortical reaction. Fusion of the gamete membranes triggers an increase of Ca2+ in the egg’s cytosol, causing cortical granules in the egg to fuse with the plasma membrane and discharge their contents. This leads to swelling of the perivitelline space, hardening of the vitelline layer, and clipping of sperm-binding receptors. The resulting fertilization envelope is the slow block to polyspermy. 5 Contact and fusion of sperm and egg membranes. A hole is made in the vitelline layer, allowing contact and fusion of the gamete plasma membranes. The membrane becomes depolarized, resulting in the fast block to polyspermy. 3 Acrosomal reaction. Hydrolytic enzymes released from the acrosome make a hole in the jelly coat, while growing actin filaments form the acrosomal process. This structure protrudes from the sperm head and penetrates the jelly coat, binding to receptors in the egg cell membrane that extend through the vitelline layer. 2 Contact. The sperm cell contacts the egg’s jelly coat, triggering exocytosis from the sperm’s acrosome. 1 Sperm-binding receptors Entry of sperm nucleus. 4

The Cortical Reaction in sea urchins Fusion of egg and sperm also initiates the cortical reaction…inducing a rise in Ca2+ that stimulates cortical granules to release their contents outside the egg A fluorescent dye that glows when it binds free Ca2+ was injected into unfertilized sea urchin eggs. After sea urchin sperm were added, researchers observed the eggs in a fluorescence microscope. EXPERIMENT RESULTS The release of Ca2+ from the endoplasmic reticulum into the cytosol at the site of sperm entry triggers the release of more and more Ca2+ in a wave that spreads to the other side of the cell. The entire process takes about 30 seconds. CONCLUSION 30 sec 20 sec 10 sec after fertilization 1 sec before Point of sperm entry Spreading wave of calcium ions 500 m

These changes cause the formation of a fertilization envelope That functions as a slow block to polyspermy

Another outcome of the sharp rise in Ca2+ in the egg’s cytosol Activation of the Egg Another outcome of the sharp rise in Ca2+ in the egg’s cytosol Is a substantial increase in the rates of cellular respiration and protein synthesis by the egg cell With these rapid changes in metabolism The egg is said to be activated

Post-Fertilization events in the sea urchin In a fertilized egg of a sea urchin (a model organism) many events occur in the activated egg Binding of sperm to egg Acrosomal reaction: plasma membrane depolarization (fast block to polyspermy) Increased intracellular calcium level Cortical reaction begins (slow block to polyspermy) Formation of fertilization envelope complete Increased intracellular pH Increased protein synthesis Fusion of egg and sperm nuclei complete Onset of DNA synthesis First cell division 1 2 3 4 6 8 10 20 30 40 50 5 60 Seconds Minutes 90

Fertilization in Mammals In mammalian fertilization, the cortical reaction Modifies the zona pellucida as a slow block to polyspermy This binding induces the acrosomal reaction, in which the sperm releases hydrolytic enzymes into the zona pellucida. 2 The sperm migrates through the coat of follicle cells and binds to receptor molecules in the zona pellucida of the egg. (Receptor molecules are not shown here.) 1 Breakdown of the zona pellucida by these enzymes allows the sperm to reach the plasma membrane of the egg. Membrane proteins of the sperm bind to receptors on the egg membrane, and the two membranes fuse. 3 The nucleus and other components of the sperm cell enter the egg. 4 Figure 47.6 Sperm nucleus Acrosomal vesicle Egg plasma membrane Zona pellucida basal body Cortical granules Follicle cell EGG CYTOPLASM Enzymes released during the cortical reaction harden the zona pellucida, which now functions as a block to polyspermy. 5

Cleavage

Cleavage Fertilization is followed by cleavage (a period of rapid cell division without growth) Cleavage partitions the cytoplasm of one large cell into many smaller cells called blastomeres (a) Fertilized egg. Shown here is the zygote shortly before the first cleavage division, surrounded by the fertilization envelope. The nucleus is visible in the center. Four-cell stage. Remnants of the mitotic spindle can be seen between the two cells that have just completed the second cleavage division. (b) Morula. After further cleavage divisions, the embryo is a multicellular ball that is still surrounded by the fertilization envelope. The blastocoel cavity has begun to form. (c) Blastula. A single layer of cells surrounds a large blastocoel cavity. Although not visible here, the fertilization envelope is still present; the embryo will soon hatch from it and begin swimming. (d)

Blastocyst formation

The polarity is defined by the distribution of yolk The eggs and zygotes of many animals, except mammals have a definite polarity The polarity is defined by the distribution of yolk With the vegetal pole having the most yolk and the animal pole having the least

Polarity The development of body axes in frogs is influenced by the polarity of the egg Anterior (a) Body axes. The three axes of the fully developed embryo, the tadpole, are shown above. Right Ventral Dorsal Left Posterior Animal hemisphere Animal pole Point of sperm entry 1 The polarity of the egg determines the anterior-posterior axis before fertilization. Vegetal hemisphere Vegetal pole Point of sperm entry 2 At fertilization, the pigmented cortex slides over the underlying cytoplasm toward the point of sperm entry. This rotation (red arrow) exposes a region of lighter-colored cytoplasm, the gray crescent, which is a marker of the dorsal side. Future dorsal side of tadpole Gray crescent First cleavage 3 The first cleavage division bisects the gray crescent. Once the anterior- posterior and dorsal-ventral axes are defined, so is the left-right axis. (b) Establishing the axes. The polarity of the egg and cortical rotation are critical in setting up the body axes.

Cleavage planes Cleavage planes usually follow a specific pattern that is relative to the animal and vegetal poles of the zygote Blastula (at least 128 cells). As cleavage continues, a fluid-filled cavity, the blastocoel, forms within the embryo. Because of unequal cell division due to the large amount of yolk in the vegetal hemisphere, the blastocoel is located in the animal hemisphere, as shown in the cross section. The SEM shows the outside of a blastula with about 4,000 cells, looking at the animal pole. Vegetal pole Blastula (cross section) Animal pole Blasto- coel 0.25 mm 0.25 mm Vegetal pole Blastula (cross section) Animal pole Blasto- coel 2-cell stage forming 4-cell stage forming Zygote 8-cell stage Eight-cell stage (viewed from the animal pole). The large amount of yolk displaces the third cleavage toward the animal pole, forming two tiers of cells. The four cells near the animal pole (closer, in this view) are smaller than the other four cells (SEM). 0.25 mm

Meroblastic cleavage Meroblastic cleavage, incomplete division of the egg (occurs in species with yolk-rich eggs, such as reptiles and birds) Epiblast Hypoblast BLASTODERM Blastocoel YOLK MASS Fertilized egg Disk of cytoplasm Zygote. Most of the cell’s volume is yolk, with a small disk of cytoplasm located at the animal pole. Four-cell stage. Early cell divisions are meroblastic (incomplete). The cleavage furrow extends through the cytoplasm but not through the yolk. Blastoderm. The many cleavage divisions produce the blastoderm, a mass of cells that rests on top of the yolk mass. Cutaway view of the blastoderm. The cells of the blastoderm are arranged in two layers, the epiblast and hypoblast, that enclose a fluid-filled cavity, the blastocoel. 3 1 2

Holoblastic cleavage, the complete division of the egg Occurs in species whose eggs have little or moderate amounts of yolk, such as sea urchins and frogs

Gastrulation

The morphogenetic process called gastrulation Rearranges the cells of a blastula into a three-layered embryo, called a gastrula, that has a primitive gut

Germ layers The three layers produced by gastrulation are called embryonic germ layers The ectoderm Forms the outer layer of the gastrula The endoderm Lines the embryonic digestive tract The mesoderm Partly fills the space between the endoderm and ectoderm

Gastrulation in a sea urchin Produces an embryo with a primitive gut and three germ layers Digestive tube (endoderm) Key Future ectoderm Future mesoderm Future endoderm Blastocoel Mesenchyme cells Vegetal plate Animal pole Vegetal pole Filopodia pulling archenteron tip Archenteron Blastopore 50 µm Mouth Ectoderm Mesenchyme: (mesoderm forms future skeleton) Anus (from blastopore) The blastula consists of a single layer of ciliated cells surrounding the blastocoel. Gastrulation begins with the migration of mesenchyme cells from the vegetal pole into the blastocoel. 1 2 The vegetal plate invaginates (buckles inward). Mesenchyme cells migrate throughout the blastocoel. Endoderm cells form the archenteron (future digestive tube). New mesenchyme cells at the tip of the tube begin to send out thin extensions (filopodia) toward the ectoderm cells of the blastocoel wall (inset, LM). 3 Contraction of these filopodia then drags the archenteron across the blastocoel. 4 Fusion of the archenteron with the blastocoel wall completes formation of the digestive tube with a mouth and an anus. The gastrula has three germ layers and is covered with cilia, which function in swimming and feeding. 5

Gastrulation in the frog More complicated than in a sea urchin SURFACE VIEW CROSS SECTION Animal pole Blastocoel Dorsal lip of blastopore Vegetal pole Blastula shrinking Archenteron remnant Ectoderm Mesoderm Endoderm Gastrula Yolk plug Key Future ectoderm Future mesoderm Future endoderm Gastrulation begins when a small indented crease, the dorsal lip of the blastopore, appears on one side of the blastula. The crease is formed by cells changing shape and pushing inward from the surface (invagination). Additional cells then roll inward over the dorsal lip (involution) and move into the interior, where they will form endoderm and mesoderm. Meanwhile, cells of the animal pole, the future ectoderm, change shape and begin spreading over the outer surface. The blastopore lip grows on both sides of the embryo, as more cells invaginate. When the sides of the lip meet, the blastopore forms a circle that becomes smaller as ectoderm spreads downward over the surface. Internally, continued involution expands the endoderm and mesoderm, and the archenteron begins to form; as a result, the blastocoel becomes smaller. 1 2 3 Late in gastrulation, the endoderm-lined archenteron has completely replaced the blastocoel and the three germ layers are in place. The circular blastopore surrounds a plug of yolk-filled cells.

Gastrulation in the chick Is affected by the large amounts of yolk in the egg Epiblast Future ectoderm Migrating cells (mesoderm) Endoderm Hypoblast YOLK Primitive streak

Organogenesis

Various regions of the three embryonic germ layers Organogenesis Various regions of the three embryonic germ layers Develop into the rudiments of organs during the process of organogenesis

Organogenesis in frog embryo Early in vertebrate organogenesis the notochord forms from mesoderm and the neural plate forms from ectoderm Neural plate formation. By the time shown here, the notochord has developed from dorsal mesoderm, and the dorsal ectoderm has thickened, forming the neural plate, in response to signals from the notochord. The neural folds are the two ridges that form the lateral edges of the neural plate. These are visible in the light micrograph of a whole embryo. Neural folds 1 mm Neural fold plate Notochord Ectoderm Mesoderm Endoderm Archenteron (a) LM

The neural plate soon curves inward forming the neural tube Formation of the neural tube. Infolding and pinching off of the neural plate generates the neural tube. Note the neural crest cells, which will migrate and give rise to numerous structures. Neural fold Neural plate Neural crest Outer layer of ectoderm Neural tube (b)

Mesoderm lateral to the notochord forms blocks called somites Lateral to the somites the mesoderm splits to form the coelom Somites. The drawing shows an embryo after completion of the neural tube. By this time, the lateral mesoderm has begun to separate into the two tissue layers that line the coelom; the somites, formed from mesoderm, flank the notochord. In the scanning electron micrograph, a side view of a whole embryo at the tail-bud stage, part of the ectoderm has been removed, revealing the somites, which will give rise to segmental structures such as vertebrae and skeletal muscle. Eye Somites Tail bud 1 mm Neural tube Notochord Neural crest Somite Archenteron (digestive cavity) Coelom (c) SEM

Organogenesis in the chick Neural tube Notochord Archenteron Lateral fold Form extraembryonic membranes YOLK Yolk stalk Somite Coelom Endoderm Mesoderm Ectoderm Yolk sac Eye Forebrain Heart Blood vessels Somites Early organogenesis. The archenteron forms when lateral folds pinch the embryo away from the yolk. The embryo remains open to the yolk, attached by the yolk stalk, about midway along its length, as shown in this cross section. The notochord, neural tube, and somites subsequently form much as they do in the frog. (a) Late organogenesis. Rudiments of most major organs have already formed in this chick embryo, which is about 56 hours old and about 2–3 mm long (LM). (b)

Structures derived from the three embryonic germ layers during organogenesis ECTODERM MESODERM ENDODERM • Epidermis of skin and its derivatives (including sweat glands, hair follicles) • Epithelial lining of mouth and rectum • Sense receptors in epidermis • Cornea and lens of eye • Nervous system • Adrenal medulla • Tooth enamel • Epithelium or pineal and pituitary glands • Notochord • Skeletal system • Muscular system • Muscular layer of stomach, intestine, etc. • Excretory system • Circulatory and lymphatic systems • Reproductive system (except germ cells) • Dermis of skin • Lining of body cavity • Adrenal cortex • Epithelial lining of digestive tract • Epithelial lining of respiratory system • Lining of urethra, urinary bladder, and reproductive system • Liver • Pancreas • Thymus • Thyroid and parathyroid glands

Developmental Adaptations of Amniotes The embryos of birds, other reptiles, and mammals Develop within a fluid-filled sac that is contained within a shell or the uterus Organisms with these adaptations are called amniotes

Extraembryonic membranes In amniotes, the three germ layers also give rise to the four extraembryonic membranes that surround the developing embryo Choroin Allantois Yolk Sac Amnion Amnion. The amnion protects the embryo in a fluid-filled cavity that prevents dehydration and cushions mechanical shock. Allantois. The allantois functions as a disposal sac for certain metabolic wastes produced by the embryo. The membrane of the allantois also functions with the chorion as a respiratory organ. Chorion. The chorion and the exchange gases between the embryo and the surrounding air. Oxygen and carbon dioxide diffuse freely across the egg’s shell. Yolk sac. The yolk sac expands over the yolk, a stockpile of nutrients stored in the egg. Blood vessels in the yolk sac membrane transport nutrients from the yolk into the embryo. Other nutrients are stored in the albumen (the “egg white”). Embryo Amniotic cavity with amniotic fluid Shell Albumen Yolk (nutrients)

Mammalian Development The eggs of placental mammals Are small and store few nutrients Exhibit holoblastic cleavage Show no obvious polarity Gastrulation and organogenesis Resemble the processes in birds and other reptiles

Early embryonic development in a human proceeds through four stages Human Development Early embryonic development in a human proceeds through four stages Endometrium (uterine lining) Inner cell mass Trophoblast Blastocoel Expanding region of trophoblast Epiblast Hypoblast Amniotic cavity Chorion (from trophoblast) Yolk sac (from hypoblast) Extraembryonic mesoderm cells (from epiblast) Amnion Chorion Ectoderm Mesoderm Endoderm Yolk sac Extraembryonic mesoderm Allantois Maternal blood vessel Blastocyst reaches uterus. 1 Blastocyst implants. 2 Extraembryonic membranes start to form and gastrulation begins. 3 Gastrulation has produced a three- layered embryo with four extraembryonic membranes. 4

Blastocyst forms at the completion of cleavage The trophoblast (the outer epithelium of the blastocyst) initiates implantation in the uterus, and the blastocyst forms a flat disk of cells As implantation is completed gastrulation begins and the extraembryonic membranes begin to form The embryonic germ layers have formed by the end of gastrulation

Morphogenesis

Morphogenesis Morphogenesis in animals involves specific changes in cell shape, position, and adhesion Morphogenesis is a major aspect of development in both plants and animals But only in animals does it involve the movement of cells

The Cytoskeleton, Cell Motility, and Convergent Extension Changes in the shape of a cell usually involve reorganization of the cytoskeleton

The formation of the neural tube is affected by microtubules and microfilaments Microtubules help elongate the cells of the neural plate. 1 Pinching off of the neural plate forms the neural tube. 4 Ectoderm Neural plate Microfilaments at the dorsal end of the cells may then contract, deforming the cells into wedge shapes. Cell wedging in the opposite direction causes the ectoderm to form a “hinge.” 2 3

The cytoskeleton also drives cell migration, or cell crawling The active movement of cells from one place to another In gastrulation, tissue invagination Is caused by changes in both cell shape and cell migration

Cell crawling is also involved in convergent extension A morphogenetic movement in which the cells of a tissue become narrower and longer Leads to extension of the cell sheet in a perpendicular direction Convergence Extension

Roles of the Extracellular Matrix and Cell Adhesion Molecules Fibers of the extracellular matrix may function as tracks, directing migrating cells along particular routes Several kinds of glycoproteins, including fibronectin promote cell migration by providing specific molecular anchorage for moving cells

Cell adhesion molecules Also contribute to cell migration and stable tissue structure One important class of cell-to-cell adhesion molecule is the cadherins

Cell Differentiation…Fate

Developmental Fate of Cells The developmental fate of cells depends on their history and on induction signals Coupled with morphogenetic changes Development also requires the timely differentiation of many kinds of cells at specific locations

Two general principles underlie differentiation during embryonic development First: During early cleavage divisions embryonic cells must somehow become different from one another Second: Once initial cell asymmetries are set up subsequent interactions among the embryonic cells influence their fate, usually by causing changes in gene expression (induction)

Fate Mapping Fate maps are general territorial diagrams of embryonic development

Fate map of frog embryo Classic studies using frogs gave indications that the lineage of cells making up the three germ layers created by gastrulation is traceable to cells in the blastula Fate map of a frog embryo. The fates of groups of cells in a frog blastula (left) were determined in part by marking different regions of the blastula surface with nontoxic dyes of various colors. The embryos were sectioned at later stages of development, such as the neural tube stage shown on the right, and the locations of the dyed cells determined. Neural tube stage (transverse section) Blastula Epidermis Central nervous system Notochord Mesoderm Endoderm (a)

Later studies developed techniques that marked an individual blastomere during cleavage and then followed it through development Cell lineage analysis in a tunicate. In lineage analysis, an individual cell is injected with a dye during cleavage, as indicated in the drawings of 64-cell embryos of a tunicate, an invertebrate chordate. The dark regions in the light micrographs of larvae correspond to the cells that developed from the two different blastomeres indicated in the drawings. (b)

Establishing Cellular Asymmetries

The Axes of the Basic Body Plan In nonamniotic vertebrates Basic instructions for establishing the body axes are set down early, during oogenesis or fertilization In amniotes, local environmental differences Play the major role in establishing initial differences between cells and, later, the body axes

Restriction of Cellular Potency In many species with cytoplasmic determinants (maternal substances in the egg that influence cell fate) Only the zygote is totipotent, capable of developing into all the cell types found in the adult Unevenly distributed cytoplasmic determinants in the egg cell Are important in establishing the body axes Set up differences in blastomeres resulting from cleavage As embryonic development proceeds the potency of cells becomes progressively more limited in all species

Blastomeres that receive half or all of the gray crescent develop into normal embryos, but a blastomere that receives none of the gray crescent gives rise to an abnormal embryo without dorsal structures. Spemann called it a “belly piece.” EXPERIMENT RESULTS CONCLUSION The totipotency of the two blastomeres normally formed during the first cleavage division depends on cytoplasmic determinants localized in the gray crescent. Left (control): Fertilized salamander eggs were allowed to divide normally, resulting in the gray crescent being evenly divided between the two blastomeres. Right (experimental): Fertilized eggs were constricted by a thread so that the first cleavage plane restricted the gray crescent to one blastomere. Gray crescent The two blastomeres were then separated and allowed to develop. Normal Belly piece 1 2

Cell Fate Determination and Pattern Formation by Inductive Signals Once embryonic cell division creates cells that differ from each other The cells begin to influence each other’s fates by induction

The “Organizer” of Spemann and Mangold Based on the results of their experiments Spemann and Mangold concluded that the dorsal lip of the blastopore functions as an organizer of the embryo

The organizer initiates a chain of inductions that results in the formation of the notochord, the neural tube, and other organs EXPERIMENT RESULTS CONCLUSION Spemann and Mangold transplanted a piece of the dorsal lip of a pigmented newt gastrula to the ventral side of the early gastrula of a nonpigmented newt. During subsequent development, the recipient embryo formed a second notochord and neural tube in the region of the transplant, and eventually most of a second embryo. Examination of the interior of the double embryo revealed that the secondary structures were formed in part from host tissue. The transplanted dorsal lip was able to induce cells in a different region of the recipient to form structures different from their normal fate. In effect, the dorsal lip “organized” the later development of an entire embryo. Pigmented gastrula (donor embryo) Dorsal lip of blastopore Nonpigmented gastrula (recipient embryo) Primary embryo Secondary (induced) embryo Primary structures: Neural tube Notochord Secondary Notochord (pigmented cells) Neural tube (mostly nonpigmented cells)

Formation of the Vertebrate Limb Inductive signals play a major role in pattern formation (The development of an animal’s spatial organization) The molecular cues that control pattern formation, called positional information Tell a cell where it is with respect to the animal’s body axes Determine how the cell and its descendents respond to future molecular signals

The wings and legs of chicks, like all vertebrate limbs begin as bumps of tissue called limb buds Anterior AER ZPA Posterior Organizer regions. Vertebrate limbs develop from protrusions called limb buds, each consisting of mesoderm cells covered by a layer of ectoderm. Two regions, termed the apical ectodermal ridge (AER, shown in this SEM) and the zone of polarizing activity (ZPA), play key organizer roles in limb pattern formation. (a) Apical ectodermal ridge 50 µm

The embryonic cells within a limb bud respond to positional information indicating location along three axes Digits Anterior Ventral Distal Proximal Dorsal Posterior Wing of chick embryo. As the bud develops into a limb, a specific pattern of tissues emerges. In the chick wing, for example, the three digits are always present in the arrangement shown here. Pattern formation requires each embryonic cell to receive some kind of positional information indicating location along the three axes of the limb. The AER and ZPA secrete molecules that help provide this information. (b)

One limb-bud organizer region is the apical ectodermal ridge (AER) (a thickened area of ectoderm at the tip of the bud) The second major limb-bud organizer region is the zone of polarizing activity (ZPA) A block of mesodermal tissue located underneath the ectoderm where the posterior side of the bud is attached to the body

Tissue transplantation experiments Support the hypothesis that the ZPA produces some sort of inductive signal that conveys positional information indicating “posterior” ZPA tissue from a donor chick embryo was transplanted under the ectoderm in the anterior margin of a recipient chick limb bud. EXPERIMENT Anterior New ZPA Donor limb bud Host limb bud ZPA Posterior In the grafted host limb bud, extra digits developed from host tissue in a mirror-image arrangement to the normal digits, which also formed (see Figure 47.26b for a diagram of a normal chick wing). RESULTS The mirror-image duplication observed in this experiment suggests that ZPA cells secrete a signal that diffuses from its source and conveys positional information indicating “posterior.” As the distance from the ZPA increases, the signal concentration decreases and hence more anterior digits develop. CONCLUSION

Signal molecules produced by inducing cells Influence gene expression in the cells that receive them Lead to differentiation and the development of particular structures